Ricin, Shiga-like toxins (SLT) and Staphylococcal enterotoxin B (SEB) have either been used as bioterrorism agents or are considered a bioterrorism threat because of their extreme toxicity and ease of administration. These toxins can be easily administered by inhalation, injection or ingestion. In the event of a mass exposure to biological toxins, identification of the agent in question is important for accurate diagnostic assessment of affected patients. It may also, however, be important to determine the fraction of the toxin which is still active; for example, if a significant fraction of the toxin is inactive, a treatment may not be as aggressive as it would be when a large fraction of the toxin is active. Distinguishing between active and inactive toxin may be advantageous because of the possibility that genetically engineered toxins, including the enzymatic portion of the toxin and a binding domain of another protein, can be used as a bioweapon agent, and may not be captured by traditional qualitative toxin detection tests.
Ricin is a highly toxic protein produced by Ricinus communis or castor bean plant. It is a category B agent, under the Biological Select Agents or Toxins, as defined by the United States Department of Human and Health Services. The major symptoms of ricin poisoning depend on the route of exposure and the dose received, though many organs may be affected in severe cases. The likely symptoms of Ricin inhalation include respiratory distress (difficulty breathing), fever, cough, nausea, and tightness in the chest. Finally, low blood pressure and respiratory failure may occur, leading to death. Swallowing of Ricin would likely lead to vomiting and diarrhea. Severe dehydration may also result. Other signs or symptoms may include seizures, and blood in the urine. Within several days, the person's liver, spleen, and kidneys might stop working, and the person could die. Ricin is unlikely to be absorbed through normal skin. Death from ricin poisoning could take place within 36 to 72 hours of exposure, depending on the route of exposure (inhalation, ingestion, or injection) and the dose received.
Shiga-like toxins (SLTs) are a class of toxins produced by pathogenic Escherichia coli strains. They cause hemolytic uremic syndrome in humans, which may lead to death.
Currently, there are no portable quantitative activity assays available for determining activity of Ricin and Shiga-like toxins. The mechanism of action of these toxins generally does not cause a break in nucleic acid phosphodiester backbone, making it difficult to determine activity. Accordingly, available assays are qualitative in nature, only determining presence or absence of these toxins in a sample, without giving any information regarding their activity. Further, the few quantitative assays that are available include tedious processes and steps. For instance, cell-free translation assays for determining activity of Ricin toxin require cell-extracts that provide transcriptional and translational molecular machinery including RNA polymerases for mRNA transcription, ribosomes for polypeptide translation, tRNA and amino acids, enzymatic cofactors and energy source, and cellular components essential for protein folding, while cytotoxicity assays for determining biological activity require bacterial or tissue culture cell. Alternatively, mass-spectrometry may be used for detecting free adenine released in a sample after Ricin attack on ribosomes, or HPLC-ESI-MS is used for detecting Ricinine, a marker of Ricin. The processes involving mass-spectrometry suffer from background noise and reduced sensitivity due to presence of interfering components in a sample; not to mention cumbersome equipment that is not portable.
Not only are these processes labor-intensive, they are also time-intensive. For instance, the rapid detection tests used by Center for Disease Control and Prevention's Laboratory Response Network take 6-8 hours, while the toxin activity tests take about 48 hours. Although there have been reports of new detection assays that only take 1-2 hours, these assays are only qualitative in nature and do not give any information on activity of the toxin. Staphylococcal enterotoxin B (SEB) is an enterotoxin produced by the bacterium Staphylococcus aureus. It is a common cause of food poisoning, with severe diarrhea, nausea and intestinal cramping often starting within a few hours of ingestion. SEB is classified as an incapacitating agent because in most cases aerosol exposure does not result in death but in a temporary, though profoundly incapacitating, illness lasting as long as 2 weeks. SEB is a superantigen, which causes massive nonspecific activation of immune system causing release of large amounts of cytokines that lead to significant inflammation.
Currently, there are no assays available for determining activity of SEB. The assays available, such as enzyme-linked immunosorbent assays (ELISA), chemiluminescence (ECL), and polymerase chain reaction (PCR), are quantitative in nature and only aid in detection of SEB.
Microfluidic systems, including “lab on a chip” or “lab on a disk” systems continue to be in development. See, Lee, B. S., et. al., “A fully automated immunoassay from whole blood on a disc,” Lab Chip 9, 1548-1555 (2009) and Madou, M. et. al., “Lab on a CD,” Annu. Rev. Biomed. Engr. 8, 601-628 (2006), which articles are hereby incorporated by reference in their entirety for any purpose.